Combined Second Harmonic Generation and Fluorescence Analyses of the Structures and Dynamics of Molecules on Lipids Using Dual-Probes: A Review.

Revealing the structures and dynamic behaviors of molecules on lipids is crucial for understanding the mechanism behind the biophysical processes, such as the preparation and application of drug delivery vesicles. Second harmonic generation (SHG) has been developed as a powerful tool to investigate the molecules on various lipid membranes, benefiting from its natural property of interface selectivity, which comes from the principle of even order nonlinear optics. Fluorescence emission, which is in principle not interface selective but varies with the chemical environment where the chromophores locate, can reveal the dynamics of molecules on lipids. In this contribution, we review some examples, which are mainly from our recent works focusing on the application of combined spectroscopic methods, i.e., SHG and two-photon fluorescence (TPF), in studying the dynamic behaviors of several dyes or drugs on lipids and surfactants. This review demonstrates that molecules with both SHG and TPF efficiencies may be used as intrinsic dual-probes in plotting a clear physical picture of their own behaviors, as well as the dynamics of other molecules, on lipid membranes.

Fluorescent and Electron-Dense Green Color Emitting Nanodiamonds for Single-Cell Correlative Microscopy.

Correlative light and electron microscopy (CLEM) is revolutionizing how cell samples are studied. CLEM provides a combination of the molecular and ultrastructural information about a cell. For the execution of CLEM experiments, multimodal fiducial landmarks are applied to precisely overlay light and electron microscopy images. Currently applied fiducials such as quantum dots and organic dye-labeled nanoparticles can be irreversibly quenched by electron beam exposure during electron microscopy. Generally, the sample is therefore investigated with a light microscope first and later with an electron microscope. A versatile fiducial landmark should offer to switch back from electron microscopy to light microscopy while preserving its fluorescent properties. Here, we evaluated green fluorescent and electron dense nanodiamonds for the execution of CLEM experiments and precisely correlated light microscopy and electron microscopy images. We demonstrated that green color emitting fluorescent nanodiamonds withstand electron beam exposure, harsh chemical treatments, heavy metal straining, and, importantly, their fluorescent properties remained intact for light microscopy.

Dual-Probe Near-Field Phaseless Antenna Measurement System on Board a UAV.

On-site antenna measurement has been recently attracting an increasing interest in order to assess the antenna performance in real operational environments. The complexity and cost of these kind of measurements have been significantly cut down due to recent developments in unmanned aerial vehicles' (UAVs) hardware and antenna measurement post-processing techniques. In particular, the introduction of positioning and geo-referring subsystems capable of providing centimeter-level accuracy together with the use of phase retrieval techniques and near-field to far-field transformation algorithms, have enabled near field measurements using UAVs. This contribution presents an improved UAV-based on-site antenna measurement system. On the one hand, the simultaneous acquisition on two measurement surfaces has been introduced and calibrated properly, thus reducing geo-referring uncertainties and flight time. On the other hand, the positioning and geo-referring subsystem has been enhanced by means of a dual-band real time kinematics (RTK) unit. The system capabilities were validated by measuring an offset reflector antenna, and the results were compared with the measurements at the spherical range in the anechoic chamber and with the measurements collected with a previous version of the implemented system.

A Red-Emitting, Multidimensional Sensor for the Simultaneous Cellular Imaging of Biothiols and Phosphate Ions.

The development of new fluorescent probes for cellular imaging is currently a very active field because of the large potential in understanding cell physiology, especially targeting anomalous behaviours due to disease. In particular, red-emitting dyes are keenly sought, as the light in this spectral region presents lower interferences and a deeper depth of penetration in tissues. In this work, we have synthesized a red-emitting, dual probe for the multiplexed intracellular detection of biothiols and phosphate ions. We have prepared a fluorogenic construct involving a silicon-substituted fluorescein for red emission. The fluorogenic reaction is selectively started by the presence of biothiols. In addition, the released fluorescent moiety undergoes an excited-state proton transfer reaction promoted by the presence of phosphate ions, which modulates its fluorescence lifetime, τ, with the total phosphate concentration. Therefore, in a multidimensional approach, the intracellular levels of biothiols and phosphate can be detected simultaneously using a single fluorophore and with spectral clearing of cell autofluorescence interferences. We have applied this concept to different cell lines, including photoreceptor cells, whose levels of biothiols are importantly altered by light irradiation and other oxidants.

Dual Probes for Positron Emission Tomography (PET) and Fluorescence Imaging (FI) of Cancer.

Dual probes that possess positron emission tomography (PET) and fluorescence imaging (FI) capabilities are precision medicine tools that can be used to improve patient care and outcomes. Detecting tumor lesions using PET, an extremely sensitive technique, coupled with fluorescence-guided surgical resection of said tumor lesions can maximize the removal of cancerous tissue. The development of novel molecular probes is important for targeting different biomarkers as every individual case of cancer has different characteristics. This short review will discuss some aspects of dual PET/FI probes and explore the recently reported examples.

Fluorescent magnetic nanoparticles for cell labeling: flux synthesis of manganite particles and novel functionalization of silica shell.

Novel synthetic approaches for the development of multimodal imaging agents with high chemical stability are demonstrated. The magnetic cores are based on La0.63Sr0.37MnO3 manganite prepared as individual grains using a flux method followed by additional thermal treatment in a protective silica shell allowing to enhance their magnetic properties. The cores are then isolated and covered de novo with a hybrid silica layer formed through the hydrolysis and polycondensation of tetraethoxysilane and a fluorescent silane synthesized from rhodamine, piperazine spacer, and 3-iodopropyltrimethoxysilane. The aminoalkyltrialkoxysilanes are strictly avoided and the resulting particles are hydrolytically stable and do not release dye. The high colloidal stability of the material and the long durability of the fluorescence are reinforced by an additional silica layer on the surface of the particles. Structural and magnetic studies of the products using XRD, TEM, and SQUID magnetometry confirm the importance of the thermal treatment and demonstrate that no mechanical treatment is required for the flux-synthesized manganite. Detailed cell viability tests show negligible or very low toxicity at concentrations at which excellent labeling is achieved. Predominant localization of nanoparticles in lysosomes is confirmed by immunofluorescence staining. Relaxometric and biological studies suggest that the functionalized nanoparticles are suitable for imaging applications.